Biotechnology

• Cell biology
• Molecular biology
• Microbial, plant and animal physiology

• Food chemistry
• Human nutritional epidemiology
• Human physiology
Useful
Understand how DNA works
microorganisms and manipulate DNA
in biotechnology:

Bacteria
Fungi
Viruses
Produce valuable Transform food products
biochemicals
 Bacteria

Bacterial enzymes, amino Bacteria are key participants Bacteria also are the most
acids, vitamins, and in gene cloning and other frequent cause of food
polysaccharides can be directly molecular biological borne illnesses.
used in food processing. procedures.
(Johnson-Green, 2002)
It is essential to have an orderly
understanding of bacterial
taxonomy
- to fully exploit their
biotechnological potential
- to avoid from undesirable effects
on food safety
Bacteria should be identified.
 What are the advanatges of bacteria in biotechnology ?

Flow of genetic
information, DNA Bacteria have single Gene expression is more
replication, transcription, chromosome with few easily modified in
and translation are less associated proteins. bacteria.
complex in bacteria.

Plasmids, the small

Plasmids can be used as
circular DNA molecules,
DNA vectors for gene
are also common in
cloning.
bacteria.
Fungi

Grow by breaking down organic

Large range of physiologically compounds and utilizing the
and structurally diverse products of this decomposition
eukaryotic organisms. as a source of carbon and
energy.

Some of them can be directly

Unicellular fungi are generally
eaten, some have the ability to
referred to as yeasts.
transform foods.
(Johnson-Green, 2002)
(Johnson-Green, 2002)
 Yeasts in Biotechnology
Widely used in gene cloning and
especially when aiming to produce Being physiologically and
large quantities of recombinant genetically more complex,
proteins. getting DNA into them is more
Frequently chosen for the expression difficult.
of mammalian proteins rather than Fewer plasmid vectors are
bacteria, as yeasts are eukaryotic. available.
 Viruses

Two basic components:

- nucleic acid (single- or double-stranded RNA or DNA)
- protein coat, the capsid

Used in cloning projects where the prime object is to move specific

segments of DNA from one organism to another
 1) Virus attaches to host cell usually binding to specific
proteins called receptors on the host cell membrane.

2) Virus enters the cell, after entry the capsid is

breakdown and viral nucleic acid is released into
cytoplasm or in case of bacteriophages, which are
bacterial viruses, only the viral genetic material enters
the cell often through endocytosis.
Lytic Cycle
3) Virus takes over the cell, normal cellular metabolism
is disrupted, and cellular enzymes, ribosomes and
metabolites are used to synthesize new viral proteins
and nucleic acids that will be assembled to make new
viral particles.

4) New virus particles are released from the cell.

Bacteriophages are released through lysis of the cell
while in eukaryotic cells releasing can be sometimes
also through budding in this case it may not be lethal
to the cell.
THE HEART OF BIOLOGY

DNA & RNA

Structural Features of DNA

Deoxyribose Nitrogenous Bases:

monosaccharides- adenine (A), guanine
Double helix
phosphate bridges (G), cytosine (C), or
backbone thymine (T)

Hydrogen bonding
between base pairs can A= T 3 linear bases
form a stable double CΞ G -Triplet codon
helix
3ʹ and 5ʹ
Notation
UNWINDING OF DNA – MELTING- DENATURATION

DNA
Transcription
Replication
 What causes DNA to unwind

ENZYME ACTIVITY INCREASE IN INCREASE IN PH

TEMPERATURE
• Re-annealing

• Primers

• Hybridization
• DNA-DNA
• RNA-RNA
• DNA-RNA
DNA Replication DNA helicase
Replication fork
RNA primer
DNA primase
DNA polymerase III
Semi-conservative replication
Leading strand
Lagging strand
Okazaki fragments
DNA strand is always
synthesized from the 5ʹ to the
3ʹ end.
Origin of replication
Transcription
of mRNA
3 STEPS OF TRANSCRIPTION
• INITIATION
• ELONGATION
• TERMINATION
 RNA Promoter
polymerase region
 Negative Regulation

In Operator region
prokaryotes Repressor

Inducer
 Positive Regulation
In
Activator-binding sites
prokaryotes
Enhancers
 RNA polymerase
In
eukaryotes General transcription
factors (GTFs)
Editing of RNA in eukaryotes

• Spliceosome: RNA+ Protein

 • Translation describes the process of protein synthesis by
ribosomes, using a mRNA transcript

• The combined processes of transcription and translation lead

to the expression of proteins
Translation
of mRNA
into Proteins
Initiation
• AUG start codon
• Ribosome-binding site (RBS)
(Shine–Dalgarno sequence)
• Transfer RNA (tRNA)
Elongation
 UAG

Termination UAA

UGA
Prokaryotic and
Eukaryotic
Ribosome
Posttranslational Processing
of Polypeptides
• Molecular chaperons
• Secreted proteins
• Signal sequence
• N-terminal-C terminal
Travel of Secreted
Protein
• ER
• -Transport vesicles
• Golgi
• -Secretory vesicles
• Membrane
 Will the promoters, termination
signals, ribosome-binding sites, and
signal sequences work in the new cell?
Relevance of
Will the RNA transcript be correctly
DNA to edited before translation?
Biotechnology
Will the new cell have the machinery
for proper posttranslational
processing?
WORKING WITH DNA
1- Purification of Nucleic Acids

• This technique is utilized to separate DNA from other cellular components.

Ø It is the process to decrease and eliminate the contamination from the nucleic acid samples.
(GC Biotech, n.d.)
Two reasons to use Nucleic Acid
Purification:

• When DNA and RNA are isolated, they should be purer.

• Specific molecular weight of DNA is needed for downstream
applications.
How is this technique performed?
First, the cell must be lysed (broken). It can be easily
performed for animal cells.

• It is made with the help of usage of solute, like

sucrose, membrane solubilizing agents e.g. Sodium
dodecyl sulfate (SDS), and proteases.
For the cells that have cell wall (Fungal, plant,
and bacterial cells):
For instance, yeast cells are mostly treated with the cell-
wall degrading enzymes to produce sphaeroblasts
(cells having only one membrane), which provides it to
break easily.

Then, a chemical, like phenol, is added for denaturation

of any protein present, and to solubilize lipids, organic
solvents are used, such as chloroform.
Afterward, the final mixture is centrifuged, and three different layers are
obtained: one involving phenol, one aqueous layer, and a chloroform
layer. After removing the aqueous layer carefully, cold ethanol is added
to precipitate DNA and RNA so that they can be avoided to be pelleted
through centrifugation and resuspended in a small amount of water or
buffer.

This kind of DNA samples are generally considered with RNAse,

degrades RNA, to prevent from contaminating RNA.

Ø RNA samples should be handled very carefully! Using gloves and

water that involves chemical inhibitors (in buffers, etc) can prevent
from contamination.
 Figure 1

2- Gel Electrophoresis

WHY IS IT USED?
q To separate fragments of different
lengths.
q To determine the length of DNA
fragments

Retrieved from: https://regi.tankonyvtar.hu/hu/tartalom/tamop412A/2011-0073_introduction_practical_biochemistry/ch10s06.html

 Figure 2
In this technique, Agarose or
polyacrylamide gel, which is
suspended in a buffer, is utilized
to see and analyze the results.
Agarose gel serves as a
conductive and stable medium
for DNA fragment migration and
separation.

HOW DOES IT WORK?

DNA is loaded into small slots
cut into the gel. Then, by using
the electrical current and the
charge that DNA carries, DNA
fragments will move the positive
pole. (From cathode to anode)
Retrieved from: https://whatisdna.net/wp-content/uploads/2017/04/dna-and-gel-electrophoresis-6-638.jpg
The fragments do not move through the pores of the gel at equal rates.

Small fragments flow much more quickly, and large fragments flow
more slowly.

Therefore, fragments that have different size can separate.

With the help of creating discrete lanes, number of samples can be

included to each gel. So, it provides a measurement of DNA fragment
length in the other lanes.
After completing the electrophoresis, ethidium bromide is added to see the DNA
in the gel, and we can observe the sample results with the help of UV light.

Figure 3
Retrieved from: https://www.researchgate.net/figure/Pulsed-field-gel-electrophoresis-Lanes-1-8-and-15-size-markers-10-and-12-isolates_fig2_8096270
DNA size can be measured in units of base pairs (bp) or kilobase pair
(kbp).

Also, another techniques should be used if the size is above the 40 kbp
because gel electrophoresis can separate and analyze small fragments of
DNA.
Agarose gel electrophoresis

Retrieved from: https://gifs.com/gif/performing-agarose-gel-electrophoresis-edvotek-video-tutorial-o2gAnK

3- Blotting and Hybridization

Øused to detect sequences of DNA.

Southern Blotting:
Þ method that is used to detect whether a specific DNA is present in the
sample or not.
Þ generally used after the gel electrophoresis
Blotting
Blotting means that the wicking of DNA from a gel or other substance
onto a membranous filter.
PROCEDURE:
Þ Gel is immersed in a solution and the paper towels are placed so that
water can flow. By doing this, DNA also flows with the water,
however, with the help of membrane filter, it is trapped on the filter.
Þ Then, DNA is put in a solution that involves NaOH and denaturated
into single strands.
Þ After the removal of NaOH, the probe (single-stranded sequence of
nucleic acids that is labeled) is added.
 Figure 4

Þ When a sequence in the

DNA sample is
complementary to the
probe, there will be a
hybridization.

Perry, J. G. (2002). Introduction to Food Biotechnology. CRC Press LCC, 2000 N.W. Corporate Blvd., Boca Raton, Florida 33431.
Þ Then the unhybridized probe is washed from the filter, and the
hybridized ones can be determined.
Þ The applied procedure is also be valid for mRNA transcripts, it is
known as Northern hybridization.

Þ RNA and DNA probes can be used in both hybridizations,

Northern and Southern.

Þ Also, without using gel electrophoresis, hybridization can occur. (e.g.

dot blots)
4- DNA Sequencing

Two methods can be used:

Þ Maxam-Gilbert Method
Þ Sanger Dideoxy Method

*Since Sanger Dideoxy Method is

more commonly utilized, we will talk
about this method for this topic.

Retrieved from: https://www.jax.org/news-and-insights/jax-blog/2017/january/bridging-

the-gaps-in-dna-sequencing
SANGER-DIDEOXY METHOD: based on the selective
incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA
replication.

§ Sample of DNA fragments, includes the desired gene, is obtained.

§ With the help of four tubes, this sample is separated.

§ For each tube, nucleotides of four bases and a synthetic analogue are
added. (such as, one tube containing guanidine triphosphate (GTP),
CTP, TTP, ATP, and dideoxy ATP)

§ And with the help of blotting, all of the nucleotides are labeled to
allow radioactivity.
§ DNA polymerase and DNA primers are also added to create a new
strand of DNA, to each tube.

§ On the other hand, it cannot add dideoxy ATP, CTP, TTP, or GTP onto
a strand. So, when DNA polymerase is forced to use a dideoxy
nucleotide, strand elongation will stop.
§ Thus, dideoxy ATP, in a series of fragments in this tube, each
providing to termination of strand elongation.

§ Then, by using polyacrylamide gel, the fragments in each tube are

separated by high-resolution gel.

§ The fragments can be detected by autoradiography (radioation-

sensitive films) or fluorescence.

§ Finally, the DNA sequence is read from either the top or the bottom of
the gel.
 TO
SUM
UP

Gauthier, M. G. (2007). The Sanger sequencing method in 7 steps. Retrieved from: https://www.researchgate.net/figure/The-Sanger-sequencing-method-in-7-steps-1-The-dsDNA-fragment-is-denatured-into-two_fig2_234248746
REFERENCES

Baron, S. (Ed.). (1996). Medical Microbiology (4th ed.). The University of Texas Medical Branch at Galveston.
Cohrt, K. O. (2021, February 22). A Quick Guide to Identifying Microbes: 9 Easy Methods. Bitesize Bio.
https://bitesizebio.com/36644/methods-microbial-identification/
Gauthier, M. G. (2007). The Sanger sequencing method in 7 steps. Retrieved from:
https://www.researchgate.net/figure/The-Sanger-sequencing-method-in-7-steps-1-The-dsDNA-fragment-
is-denatured-into-two_fig2_234248746
GC Biotech. (n.d.). Nucleic Acid Purification. Retrieved from: https://gcbiotech.com/nucleic-acid-
purification/
Julin, D. (2014). Bacteriophage and Viral Cloning Vectors. Molecular Life Sciences, 1–13.
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Perl, T. M. (2005). Pulsed-field gel electrophoresis. Retrieved from:
https://www.researchgate.net/figure/Pulsed-field-gel-electrophoresis-Lanes-1-8-and-15-size-markers-10-
and-12-isolates_fig2_8096270
Perry, J. G. (2002). Introduction to Food Biotechnology. CRC Press LCC, 2000 N.W. Corporate Blvd., Boca
Raton, Florida 33431